Reduction of T Lymphoma Cells and Immunological Invigoration in a Patient Concurrently Affected by Melanoma and Sezary Syndrome Treated With Nivolumab

Abstract

Despite the recent availability of several new drugs in hemato-oncology, T-cell lymphomas are still incurable and PD-1 blockade could represent a therapeutic chance for selected patients affected by these malignancies, although further studies are required to understand the biological effects of anti-PD-1 mAbs on neoplastic T-cells and to identify biomarkers for predicting and/or monitoring patients' response to therapy. Sezary Syndrome (SS) represents a rare and aggressive variant of cutaneous T cell lymphoma (CTCL) with a life expectancy of less than 5 years, characterized by the co-presence of neoplastic lymphocytes mainly in the blood, lymph nodes and skin. In this study we analyzed longitudinal blood samples and lesional skin biopsies of a patient concurrently affected by SS and melanoma who underwent 22 nivolumab administrations. In blood, we observed a progressive reduction of SS cell number and a raise in the percentage of normal CD4+ and CD8+ T cells and NK cells over total leukocytes. Eight weeks from the start of nivolumab, these immune cell subsets showed an increase of Ki67 proliferation index that positively correlated with their PD-1 expression. Conversely, SS cells displayed a strong reduction of Ki67 positivity despite their high PD-1 expression. On skin biopsies we observed a marked reduction of SS cells which were no more detectable at the end of therapy. We also found an increase in the percentage of normal CD4+ T cells with a concomitant decrease of that of CD8+ and CD4+ CD8+ T cells, two cell subsets that, however, acquired a cytotoxic phenotype. In summary, our study demonstrated that nivolumab marked reduced SS tumor burden and invigorated immune responses in our patient. Our data also suggest, for the first time, that Ki67 expression in circulating neoplastic and immune cell subsets, as well as an enrichment in T cells with a cytotoxic phenotype in lesional skin could be valuable markers to assess early on treatment SS patients' response to PD-1 blockade, a therapeutic strategy under clinical investigation in CTCL (ClinicalTrials.gov NCT03385226, NCT04118868).

Keywords: Ki67 proliferation index; PD-1 blockade therapy; cutaneous T-cell lymphoma; granzyme B; immune sub-populations.

Copyright © 2020 Narducci, Tosi, Frezzolini, Scala, Passarelli, Bonmassar, Monopoli, Accetturi, Cantonetti, Antonini Cappellini, De Galitiis, Rosato, Picozza, Russo and D’Atri.

Figures

FIGURE 1

Changes of circulating SS cells and immune cell subsets during nivolumab treatment. (A) Absolute counts of total CD45+ leukocytes, CD4+ T cells and SS cells were determined at T0 and the indicated weeks from the start of nivolumab, as described under section “Materials and Methods.” (B–D) PBMC were co-stained with anti-TCR-Vβ 5.1 (mix C) and anti-CD4 mAbs at T0, T42 [red arrows in graph (A)] and T60. Percentage of SS cells was evaluated in pre-gated CD4+ T cells and is showed into the plots. (E) Percentages of CD8+ T cells, NK and B cells were calculated within total CD45+ leukocytes.

FIGURE 2

PD-1 expression in SS cells and normal immune cell subsets and relative patterns of Ki67+ cell frequencies. (A) Frozen PBMCs from SS patient collected at T0 and T8 were thawed and stained for flow cytometry. Pre-gated live single SS cells, normal (n) CD4+ and CD8+ T cells, CD16+ NK cells and CD19+ B cells (see Supplementary Figure 1 for the gating strategy) were inspected for PD-1 expression by overlaying T0 vs. T8 histograms. Numbers inside plots indicate the percentage of PD-1+ cells at T0. (B) Ki67 expression patterns in the same cell subsets defined in (A). The percentage of Ki67+ cells is indicated by the numbers inside the plots. (C) The bar chart shows the T8/T0 ratios of Ki67+ cell frequencies for the indicated sub-populations.

FIGURE 3

Clinical presentation and histopathological features of SS. (A) Diffuse erythroderma involving 70% of total body at T0. (B) Reduced erythroderma and presence of vitiligo-like lesion at T8. (C–J) Hematoxylin-eosin (H&E) staining and IHC on lesional skin biopsies. (C) H&E staining of T0 biopsy revealed a dense band of atypical T lymphocytes infiltrating papillary dermis (magnification x10/0.30NA).(D) H&E staining of T18 biopsy revealed a reduced neoplastic infiltrate with a lichenoid aspect (magnification x20/0.40NA). (E–J) IHC analysis for CD3+, CD4+, and CD8+ cells showed a reduction of their density from T0 to T18 (magnification x20/0.40NA).

FIGURE 4

mIHC analysis of skin infiltrating SS cells and TILs. (A) Representative 7-color multispectral images of SS cells and TILs in lesional biopsies collected at T0, T18 and T48. Immune markers and color code are shown in the underlying legend. Original magnification X20. (B–D) Left: mIHC cell percentage of CD4+ (B), CD8+ (C), CD4+ CD8+ (D) cells calculated among total lymphocytes in biopsies collected at T0, T18 and T48. Data reported for each cell subset are the mean values and standard deviation (SD) of about 20 fields from the same sections. Right: pie charts of mIHC data from biopsies collected at T0, T18, and T48. Data reported for each cell subset are the mean values derived from the analysis of the same fields considered in the flanking histograms. (E) Representative 7-color multispectral images of SS cells and TILs in biopsies collected at T0, T18, and T48. Immune markers and color code are indicated in the underlying legend. Original magnification X20. (F,G) Pie charts of checkpoint molecule expression on CD8+ and normal CD4+ lymphocytes calculated in biopsies collected at T0, T18, and T48. Data reported for each cell subset are the mean values derived from the analysis of about 20 fields from the same sections.